A number of familial hypertrophic cardiomyopathy (FHC) causing mutations have been identified in the regulatory proteins, tropomyosin (Tm) and troponin (Tn). Most of these mutations cause an increase in the Ca2+-sensitivity of muscle contraction, i.e. the onset of force occurs at lower Ca2+ concentrations. Neither the molecular mechanisms underlying the increased Ca2+-sensitivity nor its relation to the hypertrophy of the heart are well understood. Stretch activation is another cardiac phenomenon whose molecular mechanism is not understood. The long-range goal is to understand the molecular basis of FHC and stretch activation. The main hypothesis that we will test is that both of these activations involve strongly bound cross bridges (myosin heads). We plan to: 1. Determine the contribution of the myosin head-induced vs. Ca2+-induced changes in the interactions of troponin I (TnI) with actin-Tm and with troponin C (TnC) in thin filaments reconstituted with skeletal and cardiac muscle isoforms of the regulatory proteins. The main techniques will be solution ATPase and FRET measurements. 2. Determine effects of FHC mutations on occupancy of the 3 thin filament regulatory states using equilibrium titrations and transient kinetics. Fluorescent labels on selected proteins will be used to obtain equilibrium constants and rates. 3. Determine effects of selected FHC mutations in TnI and Tm on ATPase in terms of myosin vs. Ca2+ activation. FRET measurements will be used to obtain structural information. 4. Test the hypothesis that the C-terminal domain of TnC is involved in the myosin head induced activation of the thin filament. Mutants of TnC having increased affinity for Mg2+ will be used to assess the role of divalent cation in the C-domain of TnC on thin filament function. A novel mutant of TnC which reconstitutes into the thin filament and binds Ca2+ but does not activate ATPase that was developed in this lab will be used. These experiments will lead to a better understanding of the regulatory mechanism in cardiac and skeletal muscle. In particular, a better understanding of the relative contribution of the Ca2+/troponin-dependent and the myosin S1/actin-dependent activation of the thin filament will be obtained. By identifying the protein-protein interactions that are altered in the disease state it will be possible to suggest potential targets for drug design for FHC.